The present invention relates to the field of fungal biotechnology, more particularly to a strong inducible gene expression system in fungal species, such as Rhodosporidium or Rhodotorula genus.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Rhodosporidium and Rhodotorula are two closely related genera of fungi belonging to the Pucciniomycotina subphylum of the Basidiomycota. Both are often isolated as oil or carotenoid producing yeast (1). Due to their ability to be cultured to extremely high cell density (>100 g/l dry cell mass) and accumulate more than 60% biomass as triglycerides, Rhodosporidium and Rhodotorula are fungal hosts with great biotechnological potential (2-4). Towards this goal, we have established efficient systems for genetic transformation, gene knockout and strong constitutive protein expression (5-7). However, there are no effective promoters for gene expression in Rhodosporidium and Rhodotorula to date. This becomes a major bottleneck in metabolic engineering in these hosts (8-10).
D-Amino acid oxidase (DAAO, EC 1.4.3.3) is a flavoenzyme that specifically catalyzes the oxidative deamination of D-amino acids to α-keto acids, ammonia and hydrogen peroxide (FIG. 1). DAAO activity has been widely identified, ranging from bacteria, fungi to animals [11]. DAAO is best known for its use in the production of cephalosporin, an antibiotic with an annual global market of ˜200 million US dollars (12).
The Rhodosporidium toruloides and Rhodotorula gracilisDAAO is a peroxisomal protein that has been well documented since 1987 (13). Recent studies of R. toruloides Dao1 mainly focus on its heterologous expression (14), enzyme properties (15-18) and immobilization (19-22). DAO1 mRNA transcripts have been reported to peak within 12-hr after induction with D-alanine and Dao1 protein may account for 1.0% of the soluble intracellular proteins (23), however, there is no information on how the D-amino acid inducible property of the DAO1 gene can be exploited for biotechnological applications.
Small interfering RNA (siRNA) is a class of double-stranded RNA molecules, 20-25 base pairs in length. siRNA interferes with the expression of specific genes with complementary nucleotide sequences causing mRNA to be broken down after transcription resulting in no translation. siRNAs are produced from double-stranded RNAs, e.g., from an inverted repeat of an RNA sequence and typically base-pair perfectly and induce mRNA cleavage only in a single, specific mRNA target (24). MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development. Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA except that they are expressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA which is processed, in the cell nucleus, to a stem-loop structure called a pre-miRNA by the microprocessor complex. A region of the pri-miRNA is partially self-complementary allowing the transcript to fold back onto itself to form a stem-loop structure of imperfectly dsRNA. Artificial miRNA (amiRNA) technology uses endogenous pri-miRNAs, in which the miRNA and miRNA* (passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/amiRNA* sequences that direct highly efficient RNA silencing of the targeted gene (25). miRNAs typically have incomplete base pairing to a target and inhibit the translation of multiple different mRNAs with similar sequences.
It is desired to identify inducible promoters and create nucleic acid constructs that are useful for introducing into fungal species.